Pyranose oxidase (POx) is a homotetrameric flavoprotein that catalyzes the oxidation of pyranose-configured sugars at position C-2 to corresponding 2-ketoaldoses. The wide substrate specificity makes POx potential for application in various biotechnological industries. In the present study we reported the gene cloning and heterologous expression of a POx from the basidiomycete Trametes sp. and functionally expressed the gene in Escherichia coli BL21(DE3). Based on sequence alignment, three residues were chosen for site-directed mutagenesis to obtain two single mutants (K312E and E539K) and two double mutants (T166A/E539K and K312E/E539K). In comparison to the wild-type, K312E shifted its optimal pH to 5.5 while the optimal temperature of E539K and K312E/E539K increased by 10 °C. The mutants retained more activities over broader pH ranges and higher temperatures and catalyzed d-glucose at higher efficiency (5800‒12,667 M−1 s−1 for the mutants versus 5083 M−1 s−1 for the wild-type). The recombinant POx and its mutants were all useful in gluten agglomeration and enlarging the loaf volume, which depends on the amounts of enzymes added. Interestingly, adding the same amount (0.5 nkat/g of flour) of wild-type and mutant enzymes differed in the change of loaf volumes, pinpointing that the catalytic activity is not the sole determinant in applying POx in breadmaking.
AMB Express – Springer Journals
Published: Mar 13, 2018
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